Treatment of diabetes with milk protein hydrolysate

ABSTRACT

A milk protein hydrolysate which is preferably caseinoglycomacropeptide and/or a whey protein in a bioavailable form is used for the manufacture of a composition for the treatment or prevention of diabetes or syndrome X. The invention also relates to a method of treatment or prevention of diabetes or syndrome X utilizing such compositions, a method for assessing proglucagon gene expression and GLP-1 release by a cell line derived from an adenocarcinoma of human caecum.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application is a continuation of the U.S. national stagedesignation of International application PCT/EP00/10716 filed Oct. 27,2000, the entire content of which is expressly incorporated herein byreference thereto.

FIELD OF THE INVENTION

[0002] The present invention relates to use of milk protein hydrolysatesin the manufacture of a medicament for the treatment or prevention ofdiabetes or syndrome X and a to a method of treatment of diabetes orsyndrome X which comprises administering an effective amount of acomposition comprising such milk protein hydrolysates. The presentinvention also relates to the use of sweet whey or acid whey proteins orprotein hydrolysates in the manufacture of a medicament for thetreatment or prevention of diabetes or syndrome X and to a method oftreatment or prevention of diabetes or syndrome X that comprisesadministering an effective amount of such compositions. Furthermore, thepresent invention relates to the use of caseino-glycomacropeptide(“CGMP”) in the manufacture of a medicament for the treatment orprevention of diabetes or syndrome X and to a method of treatment orprevention of diabetes or syndrome X that comprises administering aneffective amount of such compositions. In addition, the presentinvention relates to the use of NCI-H716 cells, obtained from a cellline derived from a poorly differentiated adenocarcinoma of human caecum(de Bruine et al., Virchows Archiv B Cell Pathol 62:311-320 (1992)), asa model to measure proglucagon gene expression and GLP-1 secretion.

BACKGROUND OF THE INVENTION

[0003] B. Chabance et al. (Biochimie 80, 155-165, 1998) have shown thatafter eating, many peptides derived from α-, β- or κ-caseins, includingCGMP, can be detected in stomach and blood, thus indicating that thesepeptides can cross the intestinal barrier.

[0004] Diabetes mellitus is a metabolic disorder characterized by thefailure of body tissues to store carbohydrates at the normal rate.Resistance to the action of insulin is the most important factor to typeII diabetes. When this resistance exceeds the capacity of the beta cellsto produce insulin, a person becomes diabetic. During the last 70 yearspeople suffering from diabetes have been greatly aided by receivingcontrolled amounts of insulin.

[0005] Historically, insulin has been administered by injection tocombat diabetes. Administering an injection requires expertise, andcompared to oral administration, injecting a medicament is not as safe,convenient or acceptable to the patient. In light of these concerns, itis clear that there is a need for new nutritional or therapeuticproducts that may be administered orally.

[0006] Proglucagon, synthesized by L-cells found in the distal ileum andcolon, is known to be post-translationally processed into peptidesincluding glucagon-like peptide-1 (GLP-1), a potent insulinsecretagogue. In addition to potentiating glucose-induced insulinsecretion, GLP-1 is known to stimulate proinsulin gene expression andproinsulin biosynthesis.

[0007] Other actions of GLP-1 include the inhibition of glucagonsecretion and gastric motility. GLP-1 can bind in the brain, promotingsatiety and suppressing food intake. Increasing insulin secretion is akey goal in the treatment of type II diabetes and stimulation ofendogenous release of GLP-1 is a potential/prospective alternative tointravenous administration.

[0008] Improving glucose control in diabetes can provide the advantageof reducing the associated risks of hyperglycemia, including blindness,limb amputations, kidney failure and cardiovascular disorders.

[0009] A number of in vitro cell models of animal origin have beendeveloped to study the regulation of GLP-1 secretion including a fetalrat intestinal cell culture, an isolated canine L cell, a secretin tumorcell (STC-1) cell line, and the GLUTag enteroendocrine cell line. Whilethese models have provided useful information regarding the factorswhich regulate GLP-1 secretion and proglucagon expression, they sufferfrom the problem that they do not necessarily represent the sameregulators and mechanisms which are active and occur in human L cells.

[0010] The present invention addresses the problems set out above.

SUMMARY OF THE INVENTION

[0011] Remarkably, it has now been found that a milk protein hydrolysatecan induce the release of GLP-1 and can be used to improve glucosehomeostasis in vivo.

[0012] In fact, in contrast to known studies, NCI-H716 cells have nowbeen employed, obtained from a cell line derived from a poorlydifferentiated adenocarcinoma of human caecum (de Bruine et al, VirchowsArchiv B Cell Pathol 62:311-320 (1992)). Surprisingly, the NCI-H716 cellline has now been found to be a good model for the first study ofpotential secretagogues that regulate human GLP-1 secretion in vitro. Upto now, NCI-H716 cell line was not known to be suitable for thispurpose. Nor was it previously known that any human cell line would becapable of releasing GLP-1. So far, only cell lines derived from animalswere available to serve as in vitro models to study proglucagon geneexpression and GLP-1 secretion. This property of the present cell linewill enable much simplified research on GLP-1 release.

[0013] Furthermore, the results obtained by the use of a human cell lineto conduct studies on the production or function of GLP-1 will be muchmore relevant than results derived from other animal models. In short,the NCI-H716 cell line derived from a poorly differentiatedadenocarcinoma of human caecum is likely to become a key tool forstudying proglucagon gene expression and GLP-1 secretion in humans. Thiscell line is deposited and available at the ATCC (American Type CultureCollection) under the ATCC Number CCL-251. The depositor is A. F. Gazdarand the tissue of origin is the caecum: it is derived from a colorectaladenocarcinoma.

[0014] Consequently, in a first aspect the present invention relates tothe use of a milk protein hydrolysate that is capable of inducingrelease of GLP-1 in the manufacture of a composition for the treatmentor prevention of diabetes or syndrome X.

[0015] In a second aspect, the invention provides a method of treatmentor prevention of diabetes or syndrome X which comprises administering toa person in need of such treatment an effective amount of a milk proteinhydrolysate which is capable of inducing release of GLP-1 in an amounteffective to treat or prevent diabetes or syndrome X.

[0016] In a third aspect, the present invention provides a model for thestudy of proglucagon gene expression and GLP-1 production by humanswhich includes cells obtained from a cell line derived from anadenocarcinoma of human caecum.

[0017] In a further aspect, the invention relates to a method forassessing proglucagon expression and GLP-1 release in humans byutilizing a cell line derived from an adenocarcinoma of human caecum.

[0018] Yet another aspect of the invention relates to the use of a cellline derived from an adenocarcinoma of human caecum to assessproglucagon gene expression and GLP-1 release in vitro.

[0019] An advantage of the present invention is that it provides acomposition that can be administered orally. This is both safer and moreconvenient for the patient than conventional treatment by injection ofinsulin.

[0020] Another advantage of the present invention is the reduced risk ofhypoglycemic reactions. Conventional injection of insulin carries withit the undesirable side effect of hypoglycemic reactions. The use oforal hypoglycemic agents to augment insulin secretion can also result inhypoglycemic reactions. Once the plasma glucose levels reach fastingvalues, GLP-1 no longer stimulates insulin release. The advantage ofenhancing insulin release via GLP-1 secretion is that the action ofGLP-1 is glucose dependent and therefore eliminates the risk ofhypoglycemia, i.e., the release of insulin is very fine-tuned withrespect to the blood glucose levels that are actually present.

[0021] Yet another advantage is that GLP-1 remains active in personswith diabetes whereas incretin hormone, glucose dependent insulinotropicpeptide (GIP) loses effectiveness in treating diabetes in such subjects.

[0022] Still another advantage of the present invention is that itprovides metabolic benefits in addition to the augmentation of insulinrelease. Conventional treatments raise insulin levels, but the presentinvention in addition increases insulin mRNA, increases beta-cellsensitivity, and lowers glucagon levels.

[0023] Another advantage of the present invention is that it provides acomposition that can regulate appetite and reduce food intake. Thisaction is beneficial in controlling diabetes and syndrome X.

BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Additional features and advantages of the present invention aredescribed in, and will be apparent from, the description of thepresently preferred embodiments that are set out below with reference tothe drawing figures in which:

[0025]FIG. 1 shows secretion of GLP-1 by NCI-H716 cells in response toincubation for 2 h with different doses of CGMP-Ca form.

[0026]FIG. 2 shows secretion of GLP-1 by NCI-H716 cells in response toincubation for 2 h with different doses of CGMP-Na form.

[0027]FIG. 3 shows secretion of GLP-1 by NCI-H716 cells in response toincubation for 2 h with different fractions of CGMP.

[0028]FIG. 4 shows secretion of GLP-1 by NCI-H716 cells in response toincubation for 2 h with sweet whey, acid whey and meat proteinhydrolysates, as well as with an alpha-lactose dose to measure whateffect the latter may have on the results.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0029] Within the context of this specification the word “comprises” istaken to mean “includes, among other things”. It is not intended to beconstrued as “consists of” or “only”.

[0030] Within the context of this specification the term “milk proteinhydrolysate” is taken to mean milk proteins that have been subjected toany type of hydrolysis. Thus, such milk protein hydrolysate may eveninclude intact proteins that escaped hydrolysis and also any fractionsof proteins obtained by the treatment of the hydrolysis.

[0031] Within the context of this specification the terms “sweet whey”and “acid whey” are also considered to be possible milk proteinhydrolysates, because these materials are the product of enzymatic oracid hydrolysis of milk proteins. Whey, however, as is well known in theart, can also comprise intact proteins as well as different fractions ofhydrolysed proteins.

[0032] CGMP is a milk protein hydrolysate. It is a sialylatedmacropeptide that is formed by the action of rennet or pepsin onkappa-casein from the milk of mammals. An alternative name for CGMP isk-caseinoglyco-peptide. CGMP-Ca and CGMP-Na are used as abbreviationsfor the calcium salt and sodium salt, respectively, of CGMP.

[0033] The preferred milk protein hydrolysates that are capable ofinducing release of GLP-1 comprise CGMP, or a mimetic, a homologue or afragment thereof that retains the activity of CGMP. A particularlypreferred embodiment of the milk protein hydrolysate is the calcium orsodium salts of CGMP.

[0034] Advantageously, the composition includes one or more of a sourceof carbohydrate, a source of fat and a source of protein. The preferredcompositions comprise from about 15 to about 25% protein, from about 10to about 30% fat, and from about 40 to about 60% carbohydrate.Preferably, at least a portion of the protein is provided as proteinfrom sweet whey or acid whey, while more preferably, at least a portionof the protein is provided as CGMP. Other preferred milk proteinhydrolysates which are capable of inducing release of GLP-1 compriseproteins that are present in sweet whey or acid whey.

[0035] One or more of these compositions are advantageously incorporatedinto a food formula. When this is done, the composition comprises fromabout 1 to about 50 grams, preferably from 5 to about 25 grams and morepreferably from 5 to about 10 grams CGMP per 100 g of food formula.

[0036] The composition may be administered to provide sufficient wheyprotein, CGMP or mixtures thereof to improve glucose metabolism inhumans or companion animals by increasing plasma GLP-1 levels andcontrolling glycemic response. The exact amount can be determinedwithout difficulty by administering different amounts until the correcteffect is seen. The dose of whey proteins and/or CGMP is typically fromabout 1 to about 50 grams per day, preferably from 9 to about 18 gramsper day and more preferably from 3 to about 6 grams consumed at threetimes throughout a day.

[0037] It is well known in the art how a milk or whey protein can beobtained. In general, skimmed milk is treated with enzymes or acid inorder to finally separate sweet or acid whey, respectively, which isthus deprived from the clotted casein. The sweet or acid whey thencomprises whet protein hydrolysates and also minor proteins, whichremain intact. Thus, sweet or acid whey can be obtained, e.g., as a sideproduct from the production of cheese. Although it is not necessary toadditionally process whey for use in the present invention, it isobvious to the skilled artisan that further processing is possible. Forexample, sweet and acid fluid whey can be condensed, dried, fermented,delactosed, demineralized, and deproteinated.

[0038] It is also possible to use whey concentrate or whey powder. Thelatter is especially convenient for addition to any chosen food productto produce the desired effect. It is also clear to the skilled artisanthat protein hydrolysates present in sweet or acid whey can be furtherhydrolysed, e.g., to prepare a hypoallergenic whey protein hydrolysate.According to U.S. Pat. No. 5,039,532, whey protein material may besubjected to a second hydrolysis with a proteolytic enzyme in order tohydrolyse the minor proteins remaining intact after the firsthydrolysis. Such a hydrolysate may then be used as a liquid or may bedried to a powder for incorporation into numerous food products.

[0039] CGMP may be obtained by an ion-exchange treatment of a liquidlactic raw material containing CGMP. Suitable starting materials oflactic origin may include for example, any of the following:

[0040] the product of the hydrolysis with rennet of a native caseinobtained by acidic precipitation of skimmed milk with a mineral acid oracidifying ferments, optionally with addition of calcium ions,

[0041] the hydrolysis product of a caseinate with rennet,

[0042] a sweet whey obtained after separation of casein coagulated withrennet,

[0043] a sweet whey or such a whey demineralized, for example, byelectrodialysis and/or ion exchange and/or reverse osmosis,

[0044] a concentrate of sweet whey,

[0045] a concentrate of whey proteins obtained by ultrafiltration anddiafiltration of sweet whey.

[0046] mother liquors of the crystallization of lactose from a sweetwhey, or

[0047] a permeate of ultrafiltration of a sweet whey.

[0048] A preferable method of obtaining CGMP is described, for example,in WO98/53702, and includes the decationization of the liquid rawmaterial, such that the pH is at a value of 1 to 4.5, bringing theliquid into contact with a weak anionic resin of hydrophobic matrix,predominantly in alkaline form up to a stabilized pH, and thenseparating the resin from the liquid product, followed by desorption ofCGMP from the resin.

[0049] A preferred embodiment of this composition comprises a milkprotein hydrolysate. It has been shown that skimmed milk results in CGPlevels of 1.1 μg/ml in human plasma. After yogurt ingestion, 2.8 μg/mlof CGP can be detected in the blood.

[0050] As noted above, the most preferred milk protein hydrolysatescomprise CGMP.

[0051] The composition can also contain a source of protein and at leastprotein hydrolysates from sweet whet or acid whey and/or CGMP. Dietaryprotein is preferably used as a source of protein. The dietary proteinsmay be any suitable dietary protein; for example animal protein (such asmilk protein, meat protein or egg protein); vegetable protein (such assoy protein, wheat protein, rice protein, or pea protein); a mixture offree amino acids; or a combination thereof. Milk proteins such ascasein, whey protein or soy proteins are particularly preferred.

[0052] The composition may also contain a source of carbohydrate and/ora source of fat. In a preferred embodiment of the composition, the fatsource provides about 5% to about 55% of the energy of the nutritionalformula; for example about 20% to about 50% of the energy. The lipidsmaking up the fat source may be any suitable fat or fat mixture.Vegetable fat is particularly suitable; for example soy oil, palm oil,coconut oil, safflower oil, sunflower oil, corn oil, canola oil, alongwith lecithins or the like, ether used alone or in mixtures thereof.Animal fat such as milk fat may also be added if desired.

[0053] It is also preferable to include in the composition a source ofcarbohydrate. When used, the carbohydrate provides about 40% to about80% of the energy of the nutritional composition. Any suitablecarbohydrate may be used, for example sucrose, lactose, glucose,fructose, corn syrup solids, and maltodextrins, or mixtures thereof.

[0054] It is also desirable for the composition to include dietaryfibers. When used, these can provide up to about 5% of the energy of thenutritional formula. The dietary fiber may be from any suitable origin,including for example soy, pea, oat, pectin, guar gum, gum arabic,fructooligosaccharide, or mixtures thereof.

[0055] In addition, one or more suitable vitamins and/or minerals may beincluded in the composition in amounts that can meet appropriateguidelines.

[0056] One or more food grade emulsifiers may be incorporated into thenutritional formula if desired, such as, e.g., diacetyl tartaric acidesters of mono- and di-glycerides, lecithin and mono- and di-glycerides.Similarly suitable salts and stabilizers may also be included.

[0057] A preferred embodiment of the composition is enterallyadministrable; e.g., in the form of a powder, a liquid concentrate, or aready-to-drink beverage. If it is desired to produce a powderednutritional formula, the homogenized mixture can be transferred to asuitable drying apparatus such as a spray drier or freeze drier and thenbe converted to powder.

[0058] In a further embodiment, a typical food product may be enrichedwith whey protein and/or CGMP to provide inventive compositions. Forexample, a fermented milk, a yogurt, a fresh cheese, a renneted milk, aconfectionery bar, breakfast cereal flakes or bars, drinks, milkpowders, soy-based products, non-milk fermented products or nutritionalsupplements for clinical nutrition. In these products, the amount ofwhey protein and/or CGMP that is added is at least about 0.01% byweight.

[0059] In an alternative embodiment, the composition may be incorporatedin an article of confectionery, for example a sweet (i.e., chocolate),or a sweetened beverage.

[0060] Materials and Methods

[0061] Suitable materials and methods that were followed in the examplesare now described. Materials for the cell culture:

[0062] RPMI 1640 medium, Dulbecco's Modified Eagles medium (DMEM),additives and fetal bovine serum (FBS) were from Gibco (LifeTechnologies, Basel, Switzerland). Bovine serum albumin (BSA) waspurchased from Serologicals Proteins Inc. (Kankakee, IL).

[0063] Materials for Testing the Effect of CGMP:

[0064] CGMP was obtained from R&D Konolfingen and was dissolved directlyin Krebs Ringer Buffer. Two forms of CGMP, sodium extracted and calciumextracted, were tested as well as four fractions of CGMP.

[0065] Materials for Testing the Effect of Sweet and Acid Whey:

[0066] For this study, conventional milk fractions at lab scale fromfresh bovine milk from local markets were prepared.

[0067] Rennet (pressure simple) was obtained from Rhone Poulenc Roher,Cooperation Pharmaceutique Francaise, 77000 Melun France, Batch no.1010089007, expiration date Sep. 21, 2000, 50 mg active chymosine perliter produced by TEXEL 38470 Vinay, France. CaCl₂.2H₂O, HCl 32%, aceticacid (glacial), and sodium hydroxide were also used.

[0068] Milk Fractions:

[0069] Bovine Milk Fractions

[0070] Lab scale fractions were adapted from conventional milkprocesses. Centrifugation was realized at higher acceleration rates andnon-soluble fractions were washed to increase selectivity and fractionefficacy.

[0071] Cream; Cream Washing and Skimmed Milk

[0072] Usually cream was extracted from whole milk by centrifugationbetween 3,000 and 4,500 g, and the selectivity of this step was improvedby increasing acceleration u[to 13,600 g using the fixed angle rotorSORVAL GS3 at 9,000 RPM for 30 minutes. Starting from 2,200 ml of wholemilk, 90 g of cream were recovered as a top layer.

[0073] Cream washing (3 times labeled respectively, cream 1, 2 and 3):the cream layer was dispersed in 3 water volumes (270 ml) and gentlyscattered in a bottle by manual shaking before subsequentcentrifugation.

[0074] Butter particles were spontaneously formed at the top of thebottle after the third cream washing and the buttermilk was recovered bysticking butter particles together.

[0075] A non-soluble fraction was recovered after centrifugation on thebottom of the bottles used for cream washings (Labelled: washed creamsediments).

[0076] Sweet Whey, Rennet Casein Washing and Rennet Casein

[0077] The separation of whey/casein is obtained by enzymatic treatmentof skimmed milk inducing casein clotting. 520 μl of CaCl₂ 200 mM wereadded to 520 g of skimmed milk to reach 2 mM final concentration added.The skimmed milk was heated at 35° C. then 250 μl of rennet wereimmediately added with moderate magnetic stirring. After 1 min the blendwas incubated 50 min at 35° C. in a water bath, poured into bottles forsubsequent centrifugation (13,600 g for 30 min) to separate sweet wheyfrom the non-soluble rennet casein.

[0078] Then, 476 g. of supernatant were fractionated in 10×1.3 aliquots(ependorf) and 40 ml plastic tubes. The material, labelled (Sweet whey),was frozen by immersion in liquid nitrogen and stored in a plastic bagat −20° C.

[0079] The rennet casein (45 g) was dispersed in 286 ml CaCl₂ 2 mM NaCl0.9% and centrifuged, with the supernatant aliquoted, labeled (rennetcasein washing) and then frozen in liquid nitrogen.

[0080] The 31 g of recovered rennet casein were dispersed in CaCl₂ 2 mMNaCl 0.9%, the volume was adjusted to 250 ml, and then was aliquoted,labeled (rennet casein) and frozen.

[0081] Acid Whey, Acid Casein Washing and Acid Casein

[0082] The separation of whey/casein is obtained by chemicalacidification of skimmed milk inducing casein clotting. 520 μl of CaCl₂200 mM were added to 520 g of skimmed milk to reach 2 mM finalconcentration added. The skimmed milk was acidified at 25° C. by theaddition of 32% HCl from pH 6.6 to pH 4.6 with moderate magneticstirring. After 1 min of stirring, the blend was incubated 60 min at 25°C., poured into bottles for subsequent centrifugation (13,600 g for 30min) to separate acid whey from the non-soluble acid casein.

[0083] Then, 503 g. of supernatant were fractionated in 10 X 1.3aliquots (ependorf) and 40 ml plastic tubes. The material, labelled(Acid whey), was frozen by immersion in liquid nitrogen and stored in aplastic bag at −20° C.

[0084] The acid casein (41 g) was dispersed in 233 ml sodium acetate, pH4.6, and centrifuged, with the supernatant (250 ml) aliquoted, labeled(Acid casein washing) and then frozen in liquid nitrogen.

[0085] The 28.6 g of recovered acid casein were dispersed in water, thepH was adjusted from 4.67 to 6.6 by NaOH addition and the volume wasadjusted to 250 ml, and then was aliquoted, labeled (Acid casein) andfrozen.

[0086] Cell Line and Culture Conditions:

[0087] The human NCI-H716 cells, originally developed from a poorlydifferentiated caecal adenocarcinoma, were obtained from the AmericanType Culture Collection (ATCC, Rockville, Md., USA). Cells were grown insuspension at 37° C., 5% CO₂. The culture medium consisted of RPMI 1640supplemented with 10% FBS, 2 mM L-glutamine, 100 IU/ml penicillin and100 μg/ml streptomycin. Endocrine differentiation is enhanced in vitroin NCI-H716 cells grown on an extracellular matrix (de Bruine et al,1993). Cells were, therefore, seeded in dishes coated with MATRIGEL®(Becton Dickinson, Bedford, Mass., USA) two days before experiments.

[0088] Secretion Studies:

[0089] Two days before experiments, 1×10⁶ cells were seeded in 12 wellculture plates coated with MATRIGEL®. On the day of the experiment, thesupernatant was replaced by Krebs-Ringer Bicarbonate Buffer (KRB)containing 0.2% wt/vol BSA with or without CGMP. Cells were incubatedfor 2 h at 37° C. Supernatants were collected with the addition of 50μg/ml PMSF and frozen at −80° C. for subsequent analysis byradioimmunoassay (RIA) of GLP-1. Cells were scraped with a rubberpoliceman and homogenization buffer [1 N HCl containing 5% (v/v) HCOOH,1% (v/v) trifluoroacetic acid (TFA), and 1% (v/v) NaCl] and sonicatedfor 15 s. Peptides were extracted from the cell medium and cellhomogenates using an alcohol extraction as described by the supplier ofthe GLP-1 (7-36) Total RIA Kit (Linco Research Inc., St. Charles, Mo.,USA). Concentrations of GLP-1 (Total, i.e., 7-36 amide or 9-36 amide)were measured using a commercial RIA kit (Linco Research Inc., St.Charles, Mo., USA). This kit measures GLP-1(7-36)NH₂ and GLP-1(9-36)NH2with less than 0.4% crossreactivity with GLP-1(7-37). The ED₅₀ for theassay was 72 pM. The intraassay coefficient of variance was 2.28%.

[0090] Results

[0091] CGMP Stimulates the Release of GLP-1 in the NCI-H716 IntestinalCell Line.

[0092] The amount of GLP-1 released into the medium of NCI-H716 cellstreated for 2 h with increasing concentrations (0.25-2.5 mg/ml wt/vol)of the calcium form of CGMP is shown in FIG. 1. Secretion of GLP-1 byNCI-H716 cells into the medium was normalized to the total GLP-1 content(secretion+cells) of the culture well and is expressed as a percentageof the control value. CGMP induced a dose-dependent increase in GLP-1concentrations with maximum secretion reaching 259±77% (n=minimum of 3)of the control values with 2.5 mg/ml of CGMP-Ca. The symbol * representsa significant difference from control values (p<0.05).

[0093]FIG. 2 shows the amount of GLP-1 released into the medium ofNCI-H716 cells treated for 2 h with increasing concentrations (0.25-2.5mg/ml wt/vol) of the sodium form of CGMP. Again, secretion into themedium was normalized to the total cell content of the culture well andis expressed as a percentage of the control value. CGMP induced anincrease in GLP-1 concentrations with maximum secretion reaching 255±41%(n=minimum of 3) of the control values with 2.5 mg/ml of CGMP-Ca. Thesymbol * represents a significant difference from control values(p<0.05).

[0094]FIG. 3 shows secretion of GLP-1 by NCI-H716 cells in response toincubation for 2 h with different fractions of CGMP. Secretion into themedium was normalized to the total cell content of the culture well andis expressed as a percentage of the control value. The composition ofthe different fractions was the following:

[0095] Fraction 1) Hydrolyzed CGMP, pure peptidic material, nophosphorus, no sialic acid.

[0096] Fraction 2) Hydrolyzed CGMP, high sialic acid, high phosphoruscontent. Sample is in the Na-form.

[0097] Fraction 3) CGMP fraction enriched in CMPa and CMPb, thephosphorylated compounds of CGMP. Sample is in the Na-form.

[0098] Fraction 4) CGMP fraction enriched in sialic acid. Sample is inthe Ca-form.

[0099]FIG. 3 shows that the amount of GLP-1 released into the medium byall fractions, except fraction 3 (p=0.085), was significantly increasedwith Fraction 2 resulting in the highest stimulation of 220±41% (n=3) ofthe control values. The symbol * represents a significant differencefrom control values (p<0.05).

[0100] Sweet and Acid Whey Stimulate the Release of Active GLP-1 in theNCI-H716 Intestinal Cell Line

[0101] The amount of GLP-1 released into the medium after a 2 hincubation period in the presence of 5 mg/ml milk protein hydrolysatesis shown in FIG. 4. Sweet whey and acid whey induced an increase inGLP-1 release of 298±34% and 284±21%, respectively, compared to thecontrol (p=0.03 and 0.01, respectively, n=3). This effect was not due tothe alpha-lactose content of these fractions, as the use of anequivalent alpha-lactose dose as the one contained in the differentwheys only resulted in a small increase in GLP-1 secretion (144±32%compared to the control). Moreover, another protein hydrolysate, meathydrolysate, did not induce such an effect on GLP-1 production when usedin the same amount of 5 mg/ml (132±6% compared to the control).

[0102] The GLP-1 that was secreted and reported in FIG. 4 was measureddifferently that that of FIGS. 1-3. Here, a kit was used that onlymeasures the active form of GLP-1, i.e., GLP-1 (7-37) or GLP-1 (7-36amide), but not the degraded form, GLP-1 (9-36 amide), as was includedin the data for FIGS. 1-3.

EXAMPLES

[0103] The following examples are given by way of illustration only andin no way should be construed as limiting the subject matter of thepresent application. Percentages and parts are by weight unlessotherwise indicated.

Example 1 Preparation of CGMP

[0104] Bovine sweet whey was concentrated to 17% dry matter,demineralized by electrodialysis, freed of cations on a strong cationicresin column, freed of anions on a weak anionic resin column andspray-dried in a drying tower. An analysis of the components of thiscomposition is provided in the following table: Component Percentagepresent Proteins (GMP included) 11.7 Lactose 81.7 Ash 1 Lipids 1 Waterbalance to 100

[0105] The demineralized whey powder was solubilized in deionized water.After cation removal, the solution has an initial pH of 3.8. In thepreceding plant, 392 kg of this solution was treated at the temperatureof 8° C., while stirring it in the reactor in the presence of 23 kg ofweak anionic resin of hydrophobic matrix based on polystyrene (IMAC HP661®, Rohm & Haas, regenerated in OH-form) for 4 h. Stabilization of thepH at 4.89 indicates the end of the reaction. The liquid was drawn offand the resin was recovered as above.

[0106] After concentration of the liquid to 45% dry matter byevaporation, the concentrate was spray-dried in a drying tower.

[0107] Analysis of the concentrate by HPLC showed that the reactionremoved 89% of the starting CGMP. Moreover, the powder contained 9.1% ofwhey protein, which corresponded to a yield of 90% of the whey proteins.

[0108] To recover CGMP, the resin was washed successively with deionizedwater, with 30 l of an aqueous solution at 0.5% HCl and with 30 l ofdeionized water, and the CGMP was eluted twice with 40 l of aqueoussolution at 2% Ca(OH)₂. Rinsing is carried out with 30 l of deionizedwater. After combining the eluate and rinsing volumes, the combinationwas concentrated to a volume of 25 l by ultrafiltration with a membranehaving a nominal cut-off of 3000 daltons. The retentate was freeze-driedand 900 g of CGMP were obtained, corresponding to a yield of 80%relative to the starting CGMP.

Example 2 Fermented Milk Containing CGMP or Whey Powder

[0109] A traditional fermented milk containing 1-4% fats was prepared asfollows: After standardizing whole milk, low fat milk or a mixture ofboth, 0.05% by weight of CGMP as prepared in Example 1 is added. Themixture was then pasteurized in a plate exchanger, the liquid was cooledto the fermentation temperature, a thermophilic or mesophilic lacticferment was added and incubation was carried out until a pH of <5 wasobtained.

[0110] Subsequent filling and sealing pots took place in a conventionalmanner.

[0111] Alternative embodiments having additions of 0.1%, 0.25% and 0.5%by weight of CGMPs and commercial whey powder have been prepared.

Example 3 Fermented and Gelled Milk Enriched in Probiotic BacteriaContaining CGMP or Whey Powder

[0112] Fermented and gelled milks were prepared enriched in probioticbacteria. 89.3 parts milk containing fat were mixed with 3.7 parts ofskimmed milk powder and about 0.05 by weight of CGMP as prepared inExample 1, then the mixture was preheated to 70° C. and pasteurized at92° C./6 min, and after having been cooled to 43° C. the mixture wasinoculated with 2% of a common yogurt starter comprising Streptococcusthermophilus and Lactobacillus bulgaricus and with 5% of Lactobacillusjohnsonii (La-1, CNCM I-1225). After conditioning in pots, fermentationwas carried out at 38° C. up to pH 4.6 and the pots were then cooled to6° C.

[0113] The following amounts of CGMP or commercial whey powder wereadded: 0.1%, 0.25% and 0.5% by weight.

Example 4 Fermented and Gelled Milk Enriched in Probiotic BacteriaContaining CGMP or Whey Powder

[0114] Fermented and gelled milks are prepared as described in theprevious example, wherein Lactobacillus johnsonii strain is replaced byLactobacillus acidophilus La-10 (Nestle Culture collection, Lausanne,Switzerland) (ATCC 11975).

Example 5 Enteral Composition Containing CGMP

[0115] An enteral composition with an energy density of 6.3 kJ/ml and 8%(p/v) of proteins was prepared from “low temperature” skimmed milkpowder, i.e. skimmed milk dried under controlled thermal conditions.

[0116] 20 kg of low temperature skimmed milk powder was dispersed in 100kg of demineralized water at a temperature of about 50-55° C. Thisdispersion is microfiltered by passing demineralized water through until600 kg of permeate have been eliminated. The retentate is then furtherconcentrated to around 60 kg, which represents a dry matter content of21% with a protein content, based on dry matter, of 82%.

[0117] To prepare the enteral composition, 2.3 kg of liquid retentateare mixed at 55° C. with 600 g of maltodextrin, 200 g of sucrose, 20.3 gof Tri-K citrate H2O, 9.2 g of MgCl₂6H₂O, 5.8 g of NaCl and about 0.5 to1% by weight of CGMP as prepared in Example 1.

[0118] After the ingredients were dissolved in the retentate,demineralized water is added to a total weight of the dispersion of 4.7kg. The pH was adjusted to 6.8, after which 300 g of fatty phase areintroduced, the total weight of the dispersion being 5 kg.

[0119] After homogenization and sterilization, the product had anagreeable sugary taste.

Example 6 Cereal Bar Containing CGMP

[0120] In order to prepare an expanded starting product, barley, wheat,corn or oat flour was treated in a twin-screw extruder for about 15seconds at a screw speed of about 350 RPM. in the presence ofapproximately 12% of water. After this treatment, the expanded productleft the extruder in the form of 2 to 3 mm long granules that were driedfor 20 minutes at 100° C.

[0121] The product thus obtained had a cellular structure and has thefollowing composition: Edible fibers 31% Proteins 21% Glucides 37.5%Lipids 6.5% Ash 2.4% Water 1.6%

[0122] The expanded product was incorporated in a bar intended fortreatment of diabetes, which had the following composition: Expandedproduct 39.4% Oat flakes 16.7% Sorbitol 8.4% Fructose 8.5% Apple cubes6.1% Rice crispies 4.1% Gelatin 4.0% Apricot powder 2.5% Palm oil 3.0%CGMP as 2.5% prepared in example 1 Water 4.8%

Example 7 Food Supplement Containing CGMP

[0123] A culture of the strain Lactobacillus johnsonii La-1 (CNCMI-1225) of human origin, was mixed with CGMP as prepared in example 1and spray dried according to the process given in EP0818529 so as toobtain a food supplement containing an amount of about 5% by weight ofCGMP.

[0124] The powder obtained may be used as a food supplement. A breakfastcereal, milk product or another food product may then be sprinkled withthis powder containing CGMP.

Example 8 Food Supplement Containing CGMP

[0125] A food supplement was prepared as described in example 9.However, Lactobacillus johnsonii was replaced by Lactobacillusacidophilus, La-10 (Nestec collection, Lausanne, Switzerland) or amixture of the two strains.

[0126] It should be understood that various changes and modifications tothe presently preferred embodiments described herein will be apparent tothose skilled in the art. Such changes and modifications can be madewithout departing from the spirit and scope of the present invention andwithout diminishing its attendant advantages. It is therefore intendedthat such changes and modifications be covered by the appended claims.

What is claimed is:
 1. A method of treatment of diabetes or syndrome Xwhich comprises administering to a subject in need of such treatment aneffective amount of a milk protein hydrolysate that is capable ofinducing release of GLP-1 in the subject in an amount sufficient toimprove glucose metabolism in the subject or to control glycemicresponse in the subject.
 2. The method of claim 1, wherein the milkprotein hydrolysate is a caseinoglycomacropeptide, or a mimetic,homologue or fragment thereof.
 3. The method of claim 1, wherein themilk protein hydrolysate is sweet whey, acid whey, acaseinoglycomacropeptide or a mixture thereof.
 4. The method of claim 1,wherein the milk protein hydrolysate is a calcium or sodium salt of acaseinoglycomacropeptide or a mixture thereof.
 5. The method of claim 1,wherein the hydrolysate is provided in the form of a food product, afood supplement or a nutritional supplement.
 6. The method of claim 5,wherein the hydrolysate is present in an amount of about 0.01% to about10% by weight dry matter the food product, food supplement, ornutritional supplement.
 7. The method of claim 1, wherein thehydrolysate is provided in a composition that includes one or more of afat, a carbohydrate, a dietary fiber, an emulsion, vitamins or minerals.8. The method of claim 7, wherein the hydrolysate is provided in acomposition that has a powder or liquid form.
 9. The method of claim 1,wherein the hydrolysate is included in a yogurt, a confectionery, acereal or a beverage.
 10. A method for manufacturing a composition forthe treatment or prevention of diabetes or syndrome X, which comprisesformulating the composition to induce release of GLP-1, in abioavailable form, in a subject when administered thereto, byincorporating a milk protein hydrolysate or compound including a milkprotein hydrolysate in the composition.
 11. A method for inducingrelease of GLP-1 in a subject by administering thereto a milk proteinhydrolysate or compound including a milk protein hydrolysate in anamount sufficient to cause secretion of GLP-1 in the subject.
 12. Amodel for the study of proglucagon gene expression and GLP-1 productionby humans, which includes the use of a cell line derived from anadenocarcinoma of human caecum.
 13. A model for the study of proglucagongene expression and GLP-1production by humans, which includes the use ofa cell line derived from anadenocarcinoma of human caecum.
 14. A methodfor assessing proglucagon gene expression and GLP-1 release in humanswhich comprises utilizing a cell line derived from an adenocarcinoma ofhuman caecum.
 15. The method of claim 14, wherein the cell line isNCI-H716 cell line having the ATCC number CCL-251